Purification And CDNA Cloning Of Antibacterial Peptides From The Chinese Forest Frog, Rana Chensinensis

Antimicrobial peptides is a kind of small MW polypeptide which have the antimicrobial activity.They are encoded by some special genes and exist in the innate immunity systerm of many organism.They widely exist in insect, bacteria, fungus, virus, amphibian, crustacean,mollusk, pisces, birds, plants, mammal and so on.Antimicrobial peptides are payed more attention for its small MW ,thermostability,and antimicrobial activity.Amphibian's skin expose in the outside for a long time,during the long term natural selection,it gradually form a series of defense mechanism against invading dys-factors.They are called as a natural treasurehouse of antimicrobial because of a lot of antimicrobial peptides in the skin secretion.Nowadays,people have segregated many antimicrobial peptides from Xenopus, Bombina, Uperoleia, Pseudis, Rana, Kassina, Litoria, Phyllomedusa,all most 8 genus 40 species amphibian,since 1987 Zasloff segregated the Magainins from the skin secretion of Xenopus laevis.There are 5 species (B.maxima,B.orientalis,R.nigromaculata,R.rugosa,R.chensinensis) in china,but in present,the study about the gene seque-nce of R.chensniensis has not been reported.In this study,we have segregated 3 peptides from the skin secretion of R.chensiensis by reverse-phase HPLC.The result indicated the number 2 and number 3 peptides have the antimicrobial activity by antimicrobial analysis. The result of Tris-Tricine SDS-PAGE detect their MW is about 7.0Kda,after trarsmembrane in PVDF,sequencing their amino acids sequence of N-terminatio by Edaman degradation.Then we desig-n a degeneracy primer by CODEHOP(Consensus-degenerate hybrid oligonucleotide primers),and extract the total RNA from the skin of R..chensinensis .two antimicrobial peptides cDNA gene were successfully isolated from R.chensiensis,and named as CAMPs-1(GenBanK accession number:DQ471289),CAMPs-2(GenBanK accession number: DQ471290) with 285bp and 279bp respective. CAMPs-1 encodes a predicted polypeptide of 61 amino acids with 7.103 kDa and p/4.19 and contains a 183bp open reading frame(ORP) and a 102bp 3' UTR; CAMPs-2 encodes a predicted polypeptide of 59 amino acids with 6.980 kDa and pI 4.79 and contains a 177bp open reading frame(ORF) and a 102bp 3' UTR. Bioinformatics analysis indicated that they contain a conservation sequence which compose by 22 amino acids.Phylogenetic tree analysis of CAMPs-1 and CAMPs-2 showed that it share greater similarity with the other Rana antimicrobial peptides(63%~86%,57%~81% respectively).Thus it was indicated that chensirin-1 and chensirin-2...